The numbers of SIB repeats vary between species
The QUAD1 repeats were initially identified as four highly homologous sequences located in three separate intergenic regions of the E. coli
chromosome (Rudd, 1999
). Expression of the four QUAD1 RNAs was reported in separate studies (Argaman et al., 2001
; Rivas et al., 2001
; Wassarman et al., 2001
). Since most sRNAs of E. coli
are unique sequences, we sought to determine the function of these duplicated sRNA genes. First, we reexamined the number of QUAD1 repeats encoded by E. coli
. A revision of the MG1655 sequence (Hayashi et al., 2006
) revealed a 375-nucleotide region had been omitted in the yqiK-rfaE
intergenic region, where the QUAD1d sequence is located. A fifth sequence homologous to the QUADs was identified in this region (). As there are five of these repeat sequences in MG1655, the original name QUAD1 is a misnomer. We propose to rename these sequences SIBs for s
undant sequences with the individual repeats designated as SIBa, SIBb, SIBc, SIBd, and SIBe following the original QUAD1a, QUAD1b, QUAD1c and QUAD1d nomenclature.
Sib gene homology and genetic organization
Upon searching for homologous sequences in other E. coli strains, we noted that the number of SIB repeats varies between strains (). Two sequenced EHEC strains, E. coli 0157:H7 EDL933 and E. coli 0157:H7 VT-2 Sakai, contain seven SIB repeats, with one additional repeat in the yegL-mdtA intergenic region and the other additional repeat in the ygfA-serA region. On the other hand, the genomes of E. coli CFT073 and other UPEC/APEC strains are predicted to have only four SIB repeats, with a single repeat in the yqiK-rfaE region. It is worth noting though that the “missing” sib genes of UPEC/APEC strains occur in the yqiK-rfaE intergenic region, the same region where the original sequencing error occurred in MG1655. The genomes of Shigella, Salmonella and Citrobacter also contain SIB repeats of varying numbers (). Repeats in the same intergenic region are more homologous to each other than with the repeats in other regions indicating that they are likely to be more recent duplications.
Number of sib genes in various enteric strains
An alignment of the SIB repeats shows that specific nucleotides are absolutely conserved among the enteric species (). Using the M-fold program to predict the secondary structures of the encoded RNAs, many of the conserved nucleotides are predicted to be in stem structures that are conserved across all strains (Fig. S1). Thus, there is substantial sequence and potentially structural conservation for all Sib RNAs.
All five sib genes in E. coli MG1655 are expressed
Northern analysis was carried out to determine whether E. coli MG1655 expressed all five predicted Sib RNAs. To verify that the five oligonucleotide probes were specific to a given sib gene, we also probed total RNA isolated from mutant strains carrying single deletions (see below) of each of the sib genes. As shown in , transcripts specific to each of the five sib genes were detected during growth in rich as well as minimal medium though overall the levels in minimal medium tend to be higher. Two transcripts were observed per sib gene. The patterns of expression and the ratio of the shorter and longer transcripts varied to some extent; in general, the longer transcript tended to dominate for RNA isolated from stationary-phase cells. In the case of the SibD and SibE RNAs, the longer transcript was expressed more highly in most conditions. Deletion of the one or more sib genes did not appear to alter the expression pattern of the other genes (data not shown). Fusions of the sibC and sibE promoter to the lacZ reporter were also constructed. We observed high levels of β-galactosidase activity under all conditions tested, though again the levels were somewhat higher in minimal compared to rich medium (data not shown).
Expression of the sib genes
Based on the Northern analysis, each sib gene produces two distinct transcripts approximately 150 and 110 nucleotides in length. To map the ends of the two observed transcripts, we carried out 5′ and 3′ RACE analysis. These assays showed that there is a single transcriptional start site and two distinct 3′ ends for each RNA. The lengths range from 136 to 145 nucleotides for the longer transcripts and 104 to 112 nucleotides for the shorter transcripts (; Tables S1 and S2). There is no obvious terminator corresponding to either of the 3′ ends, though the end corresponding to the longer transcript is adjacent to a long predicted stem-loop. The 3′ end corresponding to the shorter transcript is in a region predicted to be single stranded so we suggest this product may be a result of processing.
Effects of sib gene deletions
We generated a series of strains carrying single sib
gene deletions and various combinations of deletions, such as ΔsibAB
, using phage recombination (Court et al., 2003
). Growth of these deletion strains was monitored in both rich and minimal medium supplemented with various carbon sources and at different temperatures. Under these conditions, there were no discernable differences between the wild type control and any of the sib
deletion strains (data not shown).
Effects of Sib RNA overproduction
To examine the effects of Sib RNA overproduction, the individual genes were placed under the control of the PBAD
promoter of plasmid pAZ3 (Kawano et al., 2007
). The wild type strain was readily transformed with each overexpression construct, and no significant growth differences were observed between strains carrying the empty vector or the plasmids with inserts, with or without the addition of the inducing agent arabinose. This was not the case when the constructs were transformed into their respective sib
gene deletion strains. For example, successful transformations of a plasmid bearing PBAD
into the ΔsibE
strain occurred only when agar plates were supplemented with 0.1% and 0.2% arabinose ( and data not shown). No colonies were obtained when cells were plated on 0.02% and 0.002% arabinose even though these concentrations could induce expression of SibE from the plasmid (data not shown). In contrast, PBAD
plasmid transformants of the same ΔsibE
strain could be obtained with and without the inducing agent ( and data not shown). This was found to be the case for every sib
deletion strain except ΔsibA
; transformants of ΔsibA
, as well as ΔsibAB
, with the PBAD
plasmid could be obtained without the addition of arabinose ( and data not shown).
Efficiency of transformation into different sib deletion strainsa
To test whether these observations were plasmid specific, sibE
was placed under control of the PLlacO-1
promoter of plasmid pBR-plac (Guillier and Gottesman, 2006
). As was the case with the PBAD
plasmids, successful transformation into ΔsibE
required high (0.1, 0.5 or 1 mM) concentrations of IPTG even though lower (less than 0.1 mM) concentrations of IPTG could induce expression of the SibE RNA (data not shown). This finding suggests that plasmid features such as the antibiotic resistance genes are not responsible for the inability to transform a specific Sib overexpression plasmid into the corresponding deletion strain.
Synthesis of toxic Ibs proteins
The need for high levels of the Sib sRNAs for plasmid transformation into the deletion strains was reminiscent of what has been reported for some plasmid addiction modules. In these cases, cells carrying plasmids encoding a stable toxin and unstable antitoxin are killed when the plasmid is lost and the antitoxin can no longer be synthesized [reviewed in (Gerdes et al., 2005
; Hayes, 2003
)]. For the well-characterized Hok-Sok plasmid addiction module of plasmid R1, the toxin is the 52-amino acid Hok protein and the antitoxin is the Sok antisense RNA [reviewed in (Gerdes and Wagner, 2007
)]. The requirement for high levels of sib
gene expression from plasmids in the deletion strains led us to hypothesize that the Sib RNAs might regulate the expression of a toxic protein. Upon closer examination of the sib
genes, we noted a conserved small ORF encoded opposite each sib
gene (). Whole genome expression analysis using tiled arrays for E. coli
showed that indeed transcripts were expressed at low levels from the strand opposite the sib
genes (data not shown). The antisense genes were predicted to encode proteins of 18–19 amino acids containing many hydrophobic residues (). The small hydrophobic proteins are also conserved in Shigella
, and when the protein sequences are used in tblastn
searches homologs can even be found in Haemophilus
Overexpression of the Ibs proteins is toxic
To determine whether the small ORFs might be toxic and thus be responsible for the plasmid transformation defect observed with the Δsib strains, we cloned the ORFs for the SIBa, SIBc and SIBe repeats together with approximately 70 nucleotides upstream of the predicted start codons behind the arabinose-inducible promoter of pAZ3. In liquid cultures with low concentrations of arabinose, we observed a cessation of growth with slow recovery for strains carrying these plasmids (data not shown). We thus propose the name ibs (induction brings stasis) for the genes, with ibsA encoded opposite sibA etc. At higher concentrations of the inducing agent, there was an irreversible stop in growth with a significant drop in the ability of the strains to form colonies (Fig. S2 and data not shown). In addition, strains carrying these plasmids were unable to growth on agar plates supplemented with 0.2 % arabinose (). To confirm that overexpression of the Ibs proteins was responsible for the lack of growth, the fourth codon of each clone was mutated to TAG. Overexpression of these mutant constructs did not impair growth in a wild type strain; however, a supF suppressor strain was still susceptible to the toxicity of the mutant proteins (). In contrast to the case with the PBAD-sibA clone, the PBAD-ibsA clone gave a phenotype that was similar to the other clones.
We propose that the toxicity of the PBAD-sibB, PBAD-sibC, PBAD-sibD, and PBAD-sibE plasmids in the respective deletion strains is due to transcription of the ibsB, ibsC, ibsD and ibsE genes encoded on the respective plasmids. Without expression of the corresponding sib gene, the cells were unable to grow. In the wild type strains, expression of the chromosomal copy of sib genes was sufficient to repress the ibs mRNA expressed from the plasmid.
Synthesis of the toxic ShoB protein
The realization that the sib-ibs
regions encoded both a small toxic protein and the corresponding antitoxin sRNA, prompted us to examine other intergenic regions encoding two RNAs. One such region is the yfhL
interval encoding the 280–320 nucleotide RyfB and 60–63 nucleotide RyfC RNAs (Kawano et al., 2005
). These two RNAs are encoded divergently on opposite strands but share 19 nucleotides of complementarity suggesting possible regulation by base pairing (). Upon examination of the ryfB
sequence, we predicted that it could encode a hydrophobic protein of 26 amino acids (). As was carried out for the IbsA, IbsC and IbsE proteins, the region corresponding to the entire RyfB RNA was cloned in pAZ3. A strain harboring this plasmid was unable to grow with arabinose ( and Fig. S2), indicating that, like IbsA, IbsC and IbsE, high levels of the 26-amino acid protein are toxic. Upon mutation of the sixth codon of the ORF to a stop codon, the strain could survive overexpression. Again, a supF
suppressor strain restored the toxicity of the mutant clone under conditions of arabinose induction (). As ryfB
encodes a s
RF, we have renamed this gene shoB
is denoted ohsC
ppression of h
ydrophobic ORF by s
RNA). Unlike the more broadly conserved ibs-sib
loci, the shoB
genes appear to be confined to E. coli
Overexpression of the ShoB protein is toxic
SibC and OhsC RNA repression of ibsC and shoB expression
The complete complementarity between the sib and ibs transcripts and the 19 nucleotides of complementarity between the ohsC and shoB transcripts led us to propose that the Sibs and OhsC RNAs repress expression of the potentially toxic Ibs and ShoB proteins by base pairing with their respective mRNAs. We examined the effects of the SibC RNA on ibsC expression by deleting the sibC promoter and monitoring the levels of the ibsC mRNA (). SibC clearly has a negative effect on ibsC transcript levels. In fact, by Northern analysis, we were only able to detect the ibsC mRNA in a sibC promoter deletion strain. The levels of the ibsC transcript were similar under the four growth conditions tested discounting the higher background for the sample isolated from cells grown to stationary phase in minimal medium. We also deleted the ohsC gene and examined shoB mRNA levels. In contrast to what we observed for the ibsC mRNA, we did not detect a difference in shoB mRNA levels in the presence or absence of the OhsC RNA (Fig. S3).
Repression of IbsC and ShoB synthesis by the SibC and OhsC RNAs
To further test OhsC RNA regulation of shoB
expression, we constructed several translational fusions of the shoB
promoter and 5′ untranslated sequence with the lacZ
reporter gene (). Multiple 5′ ends have been mapped for the ShoB transcript (Kawano et al., 2005
). When the shoB
promoter together with the longest 5′ sequence was fused to lacZ
, no β-galactosidase activity was detected (data not shown), possibly due to inhibitory secondary structures formed by the long 5′ untranslated region. We also constructed a fusion in which the sequence between the first and second mapped 5′ ends was deleted (nucleotides 2698358 to 2698399) leaving the region of complementarity (nucleotides 2698294 to 2698313). The corresponding fusion gave low, but measurable, levels of β-galactosidase activity. Under all growth conditions tested, the levels of β-galactosidase activity were more than 2-fold higher in strains deleted for ohsC
compared to the wild type strain (), consistent with the hypothesis that the OhsC RNA negatively regulates translation of the ShoB message.
SibC and OhsC RNA repression of IbsC and ShoB toxicity
Our data suggested that the Sib and OhsC RNAs repress Ibs and ShoB protein synthesis, respectively, thereby limiting the toxicity of the small hydrophobic proteins. This was tested directly by cloning the ibsC
genes behind the PBAD
promoter of a derivative of pBAD33 (Guzman et al., 1995
) and cloning the corresponding SibC and OhsC RNA genes behind the IPTG-inducible Plac
promoter on the compatible plasmid pBR-plac. The ibsC
stop codon mutation described above was also introduced into the sibC
clone to eliminate potential expression of the IbsC protein. Cells carrying both the small protein-expression plasmid and the corresponding sRNA-expression plasmid were grown to early exponential phase (OD600
≈ 0.1). The culture was split and IPTG was added to half of each culture to induce expression of the SibC and OhsC RNAs. After 30 min, arabinose was added to all cultures to induced expression of the IbsC and ShoB proteins. As shown in , induction of IbsC and ShoB led to cell stasis of the cultures to which no IPTG was added, while the cultures treated with IPTG continued to grow. Thus the SibC and OhsC RNAs, respectively, prevent the toxic effects of IbsC and ShoB overproduction. The results also show that the SibC and OhsC RNAs do not need to be encoded in cis
in order to repress IbsC and ShoB synthesis.
Reduction in membrane potential upon IbsC and ShoB overexpression
The features of the ibs
genes are very similar to the hok
gene of plasmid R1 (Gerdes and Wagner, 2007
); all encode small hydrophobic proteins that are toxic upon overexpression and whose synthesis is repressed by sRNAs that have extensive complementarity with the corresponding mRNA. The Hok protein has been suggested to form pores in the membrane given that high levels of the protein dissipate the proton motive force (Gerdes et al., 1986
). To examine whether high levels of the IbsC and ShoB proteins are having similar detrimental effects on the membrane, we tested the ability of cells to take up the dye DiBAC4
(3) [bis-(1,3-dibarbituric acid)-trimethine oxanol]. This dye enters cells, leading to increased fluorescence, upon membrane depolarization (Wickens et al., 2000
). Cells taken 0, 5 and 20 min after induction of IbsC or ShoB by the addition of arabinose were incubated with DiBAC4
(3) for 20 min and then analyzed by flow cytometry (). No changes were observed for the plasmid control pAZ3. In contrast, induction of IbsC or ShoB had rapid and dramatic effects on the membrane integrity; after 5 min of induction, 50% and 98% of cells, respectively, had depolarized membrane and after 20 min of induction 97% and 99% of all cells were depolarized.
Changes in membrane polarization upon IbsC and ShoB overproduction
Global effects of IbsC, ShoB, LdrD and TisB overexpression
Remnants of the five hok
modules have been detected on the E. coli
K-12 genome, though all appear to have degenerated with mutations and transposon insertions (Pedersen and Gerdes, 1999
). However, the four ldr
and the unique tisB
genes (Kawano et al., 2002
; Vogel et al., 2004
) all also encode small hydrophobic proteins whose synthesis is regulated by sRNAs. We confirmed that as for the 19-amino acid IbsC protein and 26-amino acid ShoB proteins, ovexpression of the 35-amino acid LdrD protein and 29-amino acid TisB protein leads to the inhibition of cell growth and reduction in colony forming units (Fig. S2). We wondered whether the shared properties of the proteins meant that overexpression causes identical changes in the cells. To address this question and also to begin explore the biological functions of these proteins, we carried out whole genome expression analysis after inducing IbsC, ShoB, LdrD or TisB expression for 20 min (the complete data set for three independent experiments is given in Table S3).
Overall, elevated levels of IbsC lead to the largest changes in gene expression; 65 genes were induced more than three-fold and 43 genes were repressed more than three-fold in three independent experiments. The numbers of genes induced and repressed by ShoB, LdrD and TisB were lower and the fold induction varied more between experiments, especially for ShoB. We do not know the cause of the variation; possibly the levels of ShoB synthesis differed between experiments due to additional levels of regulation. Nonetheless, a number of conclusions can be drawn by examining the expression of operons for which genes are induced ≥ 10-fold () or repressed ≤ 5-fold () by overexpression of at least one of the proteins. First, a common group of genes is induced by overexpression of all four proteins. Many of these genes encode stress-response or membrane proteins. Most striking among these is the soxS mRNA, which encodes a regulator of the superoxide stress response. Northern analysis confirmed that this mRNA indeed is strongly induced by IbsC overexpression as well as to a more limited extent by high levels of ShoB, LdrD and TisB (). Other genes induced by all four proteins encode tryptophanase and proteins involved in maltose transport. These genes are induced by many different stress responses (Jones and Rudd, unpublished), and several are regulated by CRP. Some strongly-responsive members of the heat shock regulon are also induced by overexpression of all of the small hydrophobic proteins.
Genes most highly induced upon IbsC, ShoB, LdrD and TisB overexpressiona
Genes most highly repressed upon IbsC, ShoB, LdrD and TisB overexpressiona
Transcripts induced by IbsC, LdrD, ShoB and TisB overproduction
Other sets of genes were only induced or repressed by overexpression of subsets of the proteins. For example, overexpression of IbsC and TisB induced many members of the Cpx regulon, such as degP
, which are part of the cell envelope stress response. We also examined degP
() and spy
expression (data not shown) by Northern analysis. While the degP
mRNA levels are clearly induced by IbsC and TisB overexpression, the levels appear to be repressed by LdrD and ShoB overexpression (although this is not reflected in the array data). Among the genes repressed by high levels of both IbsC and ShoB is the dusB-fis
operon. Some sets of genes were affected by overexpression of only one protein. The pspABCDE
operon as well as the pspG
gene, which all encode phage shock proteins, were only induced by elevated IbsC levels. This specific induction was again confirmed by Northern analysis () where we detected strong induction of the expected 700 and 2100 nucleotide bands reported for the pspABCDE
operon (Brissette et al., 1991
Together the whole genome expression analysis shows that while overexpression of IbsC, ShoB, LdrD and TisB leads to the induction of a common set of genes, the small proteins also have unique effects. It is interesting to note that many of the genes whose expression is induced encode membrane proteins or are members of the heat shock or envelope stress responses. We suggest that the induction of these genes is a downstream consequence of the changes--to the membrane or other components of the cell--caused by the small, hydrophobic proteins. We do not think the toxic effects of the proteins are exerted through the induced genes, since, for example, overexpression of IbsC is still toxic in a ΔpspABCDE ΔpspG deletion strain (data not shown). However, the finding that overexpression of some proteins induces unique genes, indicates the proteins are not all acting in an identical manner.
Elevated pspABCDE expression in absence of SibC RNA repression of ibsC
While overexpression of IbsC, ShoB, LdrD and TisB from multicopy plasmids clearly has dramatic consequences for the cell, we wondered whether expression of the proteins from the chromosome could also affect gene expression. To examine the effects of decreased SibC RNA levels and the concominant increase in ibsC mRNA levels, we probed the total RNA samples in for pspABCDE expression. As shown in , the levels of the pspABCDE transcript were clearly induced in the sibC promoter deletion strain in the LB-stationary phase culture and even more highly in the M9-exponential culture. No induction was observed for either the wild type strain or the ΔsibC deletion strain in which the ibsC gene is also deleted. Thus even low levels of the IbsC protein, which do not have detrimental effects on growth, lead to changes in gene expression.
Transcripts induced by derepressed levels of IbsC